Cytometer having fluid core stream position control

ABSTRACT

A cytometer having two or more chambers or regions in a containment structure of sheathing fluid that may be used to provide hydrodynamic focusing of another fluid having particles to be observed. The latter fluid may be a core stream which may have its lateral position in a flow or measurement channel affected by control of at least one of the parameters of the several segments of the sheathing fluid. The lateral position of the core stream may be aligned with a light source and detector for a count and observation of the particles. Electrical signals from the detector may be fed back to a processor which may control one or more parameters of the sheathing fluid in the various chambers or regions via pumps, valves, and flow and pressure sensors. This control of parameters may provide for the positioning of the core stream. This cytometer may be miniaturized.

The present application is a divisional application of U.S. patentapplication Ser. No. 10/899,607, filed Jul. 27, 2004, now U.S. Pat. No.7,242,474.

BACKGROUND

The present invention relates generally to flow cytometers. Moreparticularly, the present invention relates to flow cytometers thatsense optical properties of microscopic particles or components in aflow stream.

This invention is related to U.S. patent application Ser. No.10/225,325, by Bernard Fritz et al., filed Aug. 21, 2002, and entitled“Optical Alignment Detection System”, which is incorporated herein byreference, and this invention is related to U.S. patent application Ser.No. 10/304,773, to Aravind Padmanabhan et al., filed Nov. 26, 2002, andentitled “Portable Scattering and Fluorescence Cytometer”, which isincorporated herein by reference. This invention also is related to U.S.Pat. No. 6,549,275 B1, by Cabuz et al., issued Apr. 15, 2003, andentitled “Optical Detection System for Flow Cytometry”; U.S. Pat. No.6,597,438 B1, by Cabuz et al., issued Jul. 22, 2003, and entitled“Portable Flow Cytometer”; U.S. Pat. No. 6,382,228 B1, by Cabuz et al.,issued May 7, 2002, and entitled “Fluid Driving System for FlowCytometry”; U.S. Pat. No. 6,700,130 B2, issued Mar. 2, 2004, by Fritz,and entitled “Optical Detection System for Flow Cytometry”; and U.S.Pat. No. 6,240,944 B1, by Ohnstein et al., issued Jun. 5, 2001, andentitled “Addressable Valve Arrays for Proportional Pressure or FlowControl”; all of which are incorporated herein by reference. The term“fluid” may be used here as a generic term that includes gases andliquids as species. For instance, air, gas, water and oil are fluids.

SUMMARY

The invention is a cytometer having a mechanism for aligning a fluidcore stream in a channel.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram of a cytometer having two sheathing fluid chambersfor controlling the position of the core stream;

FIG. 2 is an end view of the light source and detector arrangementrelative to the flow channel;

FIGS. 3-5 show three different alignments of the core stream relative tothe central axis in the flow channel;

FIG. 6-9 show and end view of a containment having two, four, six andeight sheathing fluid channels, chambers, segments or regions,respectively; and

FIG. 10 reveals an illustrative example of a miniaturized housing forthe present cytometer.

DESCRIPTION

Portions and components of cytometer 10 are not drawn to scale inFIG. 1. Portions of fluid circuits 16 and 47 and many of theircomponents, along with computer/processor 20 and reservoir 23, inactuality, may be very much larger than the components of portion 41.Flow cytometry may be used to determine certain physical and chemicalproperties of microscopic biological particles or components 11 bysensing certain optical properties of those particles or components. Todo so, for instance, the particles 11 may be arranged in single fileusing hydrodynamic focusing within a sheath fluid 12, as noted inFIG. 1. The particles 11 may then be individually interrogated by alight source and detector arrangement 38 in FIGS. 1 and 2. Each particle11 may scatter a light beam 13 and produce a scatter 14 profile. Thescatter 14 profile may be identified by measuring the light intensity atdifferent scatter angles, such as with detector component 43. Component43 may be an annular shaped detector. Certain physical and/or chemicalproperties of each particle 11 may then be determined from the scatter14 profile. If no particle 11 is impinged by a light beam 13, then adetector 42 may output a signal indicating no particle present in thepath of the light beam 13.

The controlled sample fluid 15 and supporting fluids 12 may be providedby fluidic circuits 16 and 47. A fluidic containment structure 39 mayenclose hydrodynamic focusing that causes the desired particles 11 tofall into single file in a core stream 17 surrounded by a sheath fluid12. One or more light sources 44 or light source arrangements 18 mayprovide light 13 through the core stream 17, and one or more lightdetectors 42, 43, or light detector arrangements 19 may detect thescatter 14 profiles and possible fluorescence of the particles 11 of aflow stream 30. Detector 43 may be an annular detector or an array ofannular detectors. There may be a detector 42 positioned proximate tothe center of the channel 31. An arrangement 38 may have one or morelight sources 44 and/or one or more light detectors 42, 43. Arrangement38 may include a single optical device or element arrangements 18 and 19or an array of such items. A computer or processing block 20 may useoutput signals 46 via connection or line 45 from the light detectorarrangement 19 to identify and/or count selected particles 11 in thecore stream 17. FIG. 2 is a cross-section view of channel 31 at thelight source-detector arrangement 38.

Flow sensors/pressure sensors (P-S) 24, 25, 34 may be provided in-linewith each fluid prior to hydrodynamic focusing in region 32 of enclosure39. Each flow sensor 24, 25, 34 may measure the velocity of thecorresponding fluid. The flow sensors 24, 25, 34 may be thermalanemometer type flow sensors and/or microbridge type flow sensors.Pressure sensors 24, 25 and 34 may measure the pressures of therespective fluids 12 and 15.

The flow stream 30 may include a core stream 17 surrounded by a sheathfluid 12. Core stream 17 may be formed with hydrodynamic focusing withfluid 15 having particles 11 sheathed with a fluid 12, and may movealong an axis 36 of channel 31. Particles 11 may be in suspension and ina focused flow of a single file. The fluid 15 with particles 11 may beinjected from orifice 37 that may have a size between 50 and 200microns. In FIG. 2, one dimension 28 of the channel 31 may be reduced sothat the particles 11 are controlled in position in at least thatdimension. The other dimension 29 may be several times larger to reducelight reflection problems internal to the channel 31. An illustrativeexample of dimensions 28 and 29 may be 50 microns and 200 microns,respectively. Typical particles 11 may vary from 2 microns to 20microns. Maintaining an appropriate position of the particles 11 in themidst of the larger dimension 29 may be achieved with control of thesheathing fluid 12 via parameters such as pressure and or flow of thelatter.

The velocity of the sheath fluid 12 may be different than that of thecore stream 17 for a laminar flow. However, the velocity of the sheathfluid 12 and core stream 17 may remain sufficiently low to maintainlaminar flow in the flow channel 31. Fluid 12 may enter through inputport 56 into region 54 from reservoir 21 via pump/valve 26 and flowsensor/pressure sensor 24. Fluid 12 may enter through input port 57 intoregion 55 from reservoir 21 via pump/valve 27 and flow sensor/pressuresensor 25. The fluid 15 with particles 11 may have a low volume flowrate whereas the sheath fluid volume flow rate may be larger and set bythe sheath fluid 12 pressures in regions 54 and 55 of containment 39. Abarrier 49 may be a dividing wall between the channels or regions 54 and55. A difference in pressure between fluid 15 and fluid 12 may be usedto control the fluid 15 volume flow rate. The achievement ofhydrodynamic focusing may depend on a laminar flow in which fluid 15with its particles flows in central core stream that does not mix withthe sheath fluid 12. Whether a flow is laminar may be determined from aReynolds number (R_(e)). R_(e)=dρ v/η, where d is a tube diameter or adiameter equivalent of channel 31 dimensions, ρ is fluid density, v isthe mean velocity of the fluid and η is the viscosity of the fluid. WhenR_(e) is less than 2300, the flow is laminar. When R_(e) is greater than2300, then the flow may be turbulent.

As particles 11 are hydrodynamically focused, they may be subject toshear stresses which may cause the particles to have their longerdimension (if any) oriented along the axis of their flow direction. Suchshear forces may cause some particles to be somewhat elongated in thedirection of flow.

In flow cytometer 10 using optical scattering, the particles 11 may becentered on the focused optical light beam 13 in order to achieve highsignal-to-noise and accuracy in measurements. Adjustment of the locationof the fluid core stream 17 containing the particles 11 such as cellsmay be accomplished by varying the flow parameters, (e.g., velocity,pressure and the like) of the surrounding sheathing fluid 12 inindependent regions 54 and 55 during the injection process from nozzleor orifice 37 at the hydrodynamic focusing region 32. A control loop maybe established between channel 31 having an optical light emitting anddetection arrangement 38 (i.e., a light source 44 with a focused beam 13and light detectors 42 and 43) and the control of the flow parameters(i.e., pressure and/or volume via pumps/valves 26 and 27 connected tocomputer/processor 20) in the sheathing fluid 12 portions in segments orregions 54 and 55 to move the core 17 with particles 11 to or from thecenter axis 36 where the focused light beam 13 is located so as tomaximize or minimize (e.g., null) the optical light signals 13 and/or14, which in turn may send corresponding electrical signals 46 tocomputer/processor 20 along an electrical conductor 45. Flow/pressuresensors 24 and 25 may monitor the differential flow and pressure offluid 12 going into regions 54 and 55. These components may be connectedto the computer/processor 20. As needed in maintaining control of thehydrodynamic focusing of core stream 17, the pump/valve 33 andflow/pressure sensor may be connected to computer/processor 20.

A flow cytometer measurement channel 31 may consist of a core region 17of laminar flow containing the isolated particles 11 to be measuredsurrounded by a region of sheathing fluid 12. Both sheathing andparticle 11 fluid regions may be injected into the measurement channel31 by way of a hydrodynamic focusing mechanism. For a cytometer 10 whichuses optical scattering as a measurement mechanism, it may be importantto place the particles 11 substantially in the focused optical beamformed by the light source 18. This factor may be accomplished byadjusting the focused beam 13 to coincide with the particular locationof the core 17 channel 31, and this may require the use of eithermultiple optical sources or a mechanical mechanism to translate or steerthe optical beam 13. The same task may be accomplished by using asimple, fixed optical source 18/detector 19 module arrangement 38,adjusting the flow configuration to move the core stream 17, containingthe fluid 15 with particles 11, sideways in channel 31 to the locationof focused optical light beam 13. This may be done by dividing thesheathing fluid 12 into a number of independent channels, segments orregions 54 and 55, as shown in FIG. 1, at the injection nozzle ororifice 37 region of the hydrodynamic focusing device. The sheathingfluid 12 may surround the central core stream 17 and generally keep thecore stream 17 away from the walls of the measurement channel 31 andmaintain the core stream in a laminar flow. The location of core stream17 may be determined by the specific flow parameters (e.g., pressureand/or flow) of the sheathing fluid 12 segments in regions 54 and 55,respectively, and thus by varying these parameters, the core stream'slocation may be made to coincide with the focused optical light beam 13.These parameters may be controlled via pump/valve 26 and pump/valve 27by the computer/processor 20. This may be implemented with a feedbackcontrol loop between the electrical signal 46 produced by thesource-particle-detector assembly 38 and the sheathing fluid 12 flowcircuit 16. Fluid 15 may be controlled by fluid circuit 47. Componentpumps/valves 26, 27, 33, and flow/pressure sensors 24, 25, 34, may beelectrically connected to computer/processor 20 via conductors 48. Also,there may be fluid level indicators in reservoirs 21 and 22 connectedvia conductors 48 to computer/processor 20.

FIGS. 3-5 show the various alignments of flow stream 17 with axis 36 ofchannel 31. FIG. 3 shows the flow stream 17 and respective particles 11aligned with axis 36. FIG. 4 shows the flow stream 17 and respectiveparticles 11 to be off relative to axis 36 in one direction, and FIG. 5shows the flow stream 17 and respective particles 11 to be off relativeto axis 36 in the other direction. Looking down the channel 31 towardsthe discharge end, the dashed sketch of particle 11 to the left of axis36 represents the misalignment in FIG. 4, and the dashed sketch ofparticle 11 to the right of axis 36 represents the misalignment in FIG.5.

FIG. 6 shows an end view of containment 39 having two sheathing fluid 12regions or channels 54 and 56. Divider or barrier 49 may establish theseregions. The controlling sheathing fluid may have more than two regionsin containment 39, along with the corresponding additional pumps/valves,flow sensors/pressure sensors, as needed, and respective connections tocomputer/processor 20 to control the core stream 17 in channel 31. Forinstance, FIG. 7 shows containment 39 having four regions 61, 62, 63 and64 delineated by barriers 49. FIG. 8 shows the containment with sixregions 65, 66, 67, 68, 69 and 70, and FIG. 9 shows containment 39 witheight regions 71, 72, 73, 74, 75, 76, 77 and 78.

There may be a miniaturized portable version 80 of cytometer 10 providedin a housing 81 sufficiently small to be appropriately and comfortablywearable on a person. As one illustrative example in FIG. 10, thehousing 81 may be sized similar to a wrist watch. The wearable housing81 may include, for example, a base 82, a cover 83, and a fastener 84that secures the base 82 to the cover 83. The fluid drivers or pumps 26,27, 33, including regulating valves, respectively, of fluid circuits 16and 44, may be incorporated into the cover 83, while the fluidreservoirs 21, 22, 23, and flow sensors/pressure sensors (P-S) offluidic circuit 16 may be incorporated into a removable cartridge 85that is inserted into the housing 81. Core stream 17 may be illuminatedthrough channel windows 86 of the flow stream 30. The fluidic circuits47 and 16, as an illustrative example, may dilute a blood sample,perform red cell lysing, and perform hydrodynamic focusing for corestream 17 formation and control. The light source(s) 18 may be situatedin either the base 82 or the cover 83, and aligned with the flow stream17 of the removable cartridge 85. The light detector(s) 19 may beprovided generally opposite of the light source(s) 19. The processor 20and batteries may be provided in either the base 82 or the cover 83 ofthe housing 81. The light source(s) 18, light detector(s) 19, andassociated control and processing electronics 20 may performdifferentiation and counting of the particles 11, and feedback forcontrol of the core stream 17 in channel 31, based on light beam 13 andlight scattering 14 signals.

Although the invention has been described with respect to at least oneillustrative embodiment, many variations and modifications will becomeapparent to those skilled in the art upon reading the presentspecification. It is therefore the intention that the appended claims beinterpreted as broadly as possible in view of the prior art to includeall such variations and modifications.

1. An integrated fluid positioning system, comprising: a cartridgeincluding: a hydrodynamic focusing region; a channel region situateddownstream and fluidly coupled to the hydrodynamic focusing region; aplurality of sheathing fluid regions fluidly coupled to the hydrodynamicfocusing region; at least one sheathing fluid reservoir fluidly coupledto the plurality of sheathing fluid regions; a core fluid region fluidlycoupled to the hydrodynamic focusing region; a waste fluid reservoirsituated downstream and fluidly coupled to the channel region; anillumination and detection region proximate to the channel region; andwherein the plurality of sheathing fluid regions are separated by atleast one barrier that extends from a side wall in the sheathing fluidregions inward toward the core fluid region; and wherein the sheathingfluid enters each of the plurality of sheathing fluid regions throughseparate input ports.
 2. The system of claim 1, wherein a core fluidfrom the core fluid region is sheathed with a sheathing fluid from eachof the plurality of sheathing fluid regions into a core stream by thehydrodynamic focusing region.
 3. The system of claim 2, wherein: eachsheathing fluid from each of the plurality of sheathing fluid regionshas a parameter changeable by a parameter controller; and the corestream has a position that is changeable by the parameter of at leastone sheathing fluid.
 4. The system of claim 3, wherein the core streamis positioned in the illumination and detection region.
 5. The system ofclaim 4, further comprising an interface connecting at least twoparameter-controllers for positioning the core stream in theillumination and detection region.
 6. A cytometer system comprising: aflow channel; a hydrodynamic focusing channel situated upstream andfluidly connected to the flow channel; an injection channel situatedupstream and fluidly connected to the hydrodynamic focusing channel, forinjecting a sample into the hydrodynamic focusing channel; three or moresheath fluid regions situated upstream and fluidly connected to thehydrodynamic focusing channel for delivering a corresponding sheathfluid to a different part of the hydrodynamic focusing channel,collectively around the sample injected by the injection channel;wherein the three or more sheath fluid regions are circumferentiallyoriented around the hydrodynamic focusing channel and separated in partby a barrier; and wherein the sheath fluid enters each of the three ormore sheath fluid regions through separate input ports.
 7. A cytometersystem comprising: a flow channel; a hydrodynamic focusing channelsituated upstream and fluidly connected to the flow channel; aninjection channel situated upstream and fluidly connected to thehydrodynamic focusing channel, for injecting a sample into thehydrodynamic focusing channel; and two or more sheath fluid regionssituated upstream and fluidly connected to the hydrodynamic focusingchannel for delivering a corresponding sheath fluid to a different partof the hydrodynamic focusing channel, collectively around the sampleinjected by the injection channel, wherein each of the two or moresheath fluid regions are separated from adjacent sheath fluid regions byat least one barrier that extends from a side wall inward toward theinjection channel; wherein the sheath fluid enters each of the two ormore sheath fluid regions through separate input ports.